Summary: Red blood cell (RBC) alloimmunization can make it difficult to procure compatible RBCs for future transfusion, which can directly increase morbidity and mortality in transfusion-dependent individuals. While patients who develop multiple alloantibodies against distinct alloantigens are particularly challenging to manage, the immune events during initial alloimmunization that may increase the likelihood of generating additional alloantibodies following subsequent transfusion remain unknown. Our long-term goal is to identify immune factors that enhance subsequent alloimmunization events in previously alloimmunized individuals in order to prevent the accumulation of multiple alloantibodies in transfusion dependent individuals. Our central hypothesis is that initial alloimmunization events directly enhance subsequent RBC alloimmunization by inducing CD4 T cells that possess the ability to directly activate B cells against a completely unrelated RBC alloantigen following subsequent transfusion. Our hypothesis is formulated on the basis of our recent discovery that B cells specific for one antigen (the HOD (HEL, OVA and Duffy) antigen) not only internalize HOD following RBC engagement, but likewise remove and internalize additional RBC components, suggesting that B cells may possess the ability to remove multiple antigens following engagement of the target antigen. Consistent with this, adoptive transfer of CD4 T cells primed by KEL RBC transfusion in the presence of poly I:C, which induces viral-like inflammation, directly enhances alloantibody formation against the completely distinct HOD antigen following subsequent transfusion of RBCs expressing HOD and KEL. Depletion of marginal zone (MZ) B cells, a unique B cell population previously shown to be critical in the initiation of alloantibodies, inhibits KEL RBC priming and the HOD RBC boost following HOD x KEL RBC transfusion, suggesting that MZ B cells work in concert with previously recognized bridging channel 33D1+ dendritic cells (33D1+ DCs) shown to be critical in the initial activation of CD4 T cells following HOD RBC transfusion. In contrast, while KEL RBC-induced alloimmunization requires type I interferons (IFNab) and HOD RBC-induced alloimmunization requires toll-like receptor (TLR) signaling, KEL-induced alloimmunization in the presence of PIC requires both IFNab and TLRs, suggesting that while innate immune pathways may differ for KEL and HOD RBC-induced alloimmunization, PIC allows KEL RBCs to engage TLRs and prime a subsequent HOD boost. We will use a series of pre-clinical models to define the key priming and subsequent boosting pathways by testing the following specific aims: Aim 1: Define the role of MZ B cells, 33D1+ DCs, IFNab and TLRs in PIC/KEL RBC-induced priming. Aim 2: Define the role of MZ B cells, 33D1+ DCs, and TLRs in subsequent KEL-mediated HOD RBC boost. We think that successful completion of these aims will define key immunological priming and boosting events that facilitate alloimmunization and therefore will provide an important framework to develop rational approaches to prevent the development of RBC alloantibodies against multiple alloantigens in chronically transfused individuals.